This invention relates to a photoluminescent system for the detection of sample substances, as derived from biological fluids or tissue labelled with photoluminescent materials and to fluorometers adapted for accurate measure of such labelled samples on a support surface of a carrier member.
There are many techniques available for the detection of an unknown quantity of a biologically derived sample (e.g., serum or urine). During such techniques, a labelled substance which has reacted with the sample must be separated from the unreacted labelled substances which includes free and non-specifically bound substance. This separation in liquid form is known to be inefficient, unreliable, and tedious. Many solutions have been proposed to solve this problem by the use of diagnostic reagents coated on a solid surface which combine with the labelled substance.
In one technique, reagents are coated upon plastic test tubes by physical adsorption of antibodies specific to the sample substance to be tested. See, e.g., article by Catt et al in the Journal of Biochemistry, 1966, Vol. 100, page 31c and in Science, Vol. 158, page 1570, 1967. This technique is difficult to control because of the non-uniformity of the plastic surface and imprecision in the coating technique. Furthermore, during washing to remove unreacted labelled substance, a relatively weak physical coating bond holding the antibodies can be disrupted resulting in their loss along with reacted labelled substance. Also, this technique requires a separate procedure for the coating of each test tube. This would be particularly time consuming, especially to insure reproducibility, if covalent attachment were employed to prevent the loss of diagnostic reagent. In addition, such test tube coating does not lend itself to the precise viewing required in a solid front surface fluorometric system. Furthermore, use of a test tube restricts covalent attachment to the material of construction used in making the tube.
Another solid surface approach is set forth in Bratu, Jr., et al U.S. Pat. No. 3,826,619. This system employs a physically adsorbed diagnostic reagent coated on the tip of a holder. This tip is first fitted into a receptacle for the sample and then into a receptacle for the labelled substance. This system is subject to inaccuracies because of losses in rubbing of the unprotected tip against the close fitting receptacle. Also, there is no ability to stir the tip in the receptacle leading to long incubation periods. Additionally, this technique does not lend itself to reproducible mass production as each holder must be individually coated with diagnostic reagent. Furthermore, it is undesirable for precise viewing.
An improved solid surface system is described in Deindoerfer et al patent application, Ser. No. 627,941, filed Nov. 3, 1975. In a preferred embodiment, a disc bearing a fluorescently-labelled diagnostic reagent is carried by an enlongate holder and inserted into a viewing housing of a fluorometer for precise positioning of the disc. However, the disc is not essentially isolated from the remainder of the fluorometer or the environment. Therefore, if the fluorescent label on the disc is read while coated with a liquid layer, it is subject to evaporative cooling. As it is known that the intensity of a fluorescent signal is dependent on temperature, such cooling would cause a drift in signal. If the evaporation is sufficient to cause the liquid layer to become discontinuous, this produces an additional source of signal drift based upon a modification of the optical characteristics of the surface. The advantages of reading the surface while wet are set forth in the copending Bolz et al patent application, Ser. No. 690,975, entitled "Method for Reading a Wet Fluorescent Surface", filed May 28, 1976, now U.S. Pat. No. 4,025,310, and are summarized hereinafter.